Proteins can become oxidized under a variety of conditions in vitro and in vivo. Specific polypeptide changes include formation of aldehyde (carbonyl) groups, generation of methionine sulfoxide and dityrosine, rearrangement of disulfide bonds, cleavage of peptide bonds, and protein aggregation. The consequences of protein oxidative modifications are varied. Some modifications lead to differential susceptibility of a protein to proteolytic cleavage while others cause gain or loss of biological activity. For example, we have found that oxidative modification of fibrinogen inhibits ability of the protein to undergo clotting. Conversely, oxidation of plasma protease inhibitors can lead to an increase of clotting activity. Our research is designed to elucidate the conditions under which proteins acquire oxidative modifications and to ensure that the assays employed provide a correct assessment of the types of modifications incurred. In recently completed experiments, we determined that the carbohydrate moieties of glycoproteins do not undergo oxidative modification to carbonyl groups under conditions that oxidize amino acid side chains. In addition, we validated the use of a Western blot assay for protein carbonyls for use on glycoproteins. Currents studies are aimed at determining the extent to which proteins undergo oxidative modification within cells exposed to varying growth conditions, including oxidative stress. Because cellular lipids are also targets for oxidative attack and can modify proteins through conjugation reactions, it will be important to distinguish between lipid-derived protein modifications and direct protein oxidative modification. Assays to accomplish this are currently being developed.